DOCSLIB.ORG

Mycoplasma Hoechst Stain Kit

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mycoplasma Hoechst Stain Kit Catalog Number: 3030000 Mycoplasma Hoechst Stain Kit 1. Kit Contents Item Amount Catalog Number 1. Hoechst Stain (0.5 μg/ml) 10 ml 3030145 2. 10X HBSS without Phenol Red 10 ml 3030245 and Sodium Bicarbonate 3. Mounting Medium 10 ml 3030345 4. Fixed Control Slide 5/pk 3030400 NOTE: 1. Each kit contains stain sufficient for 100 tests. 2. Dilute the 10X HBSS reagent 1/10 for use with this kit. 2. Introduction Many methodologies exist which are used to isolate and identify mycoplasma contaminants. Among these are direct growth on agar, broth or semisolid media, enzymatic procedures, RNA labeling, autoradiography, and staining with DNA fluorochromes. All of the above tests with the exception of the DNA staining require time, expertise and a substantial amount of equipment and reagents. The DNA fluorochrome staining is the only known method which is sufficiently rapid and sensitive to allow frequent testing at each passage. A cell sheet between 50-80% confluent is fixed and stained with the DNA specific dye and examined under fluorescent microscopy. Non-nuclear staining will be readily apparent and contaminants will stand out boldly against a black background. The nature of the contaminant may be determined by its morphology, size and relationship to the cells. Several DNA fluorochromes such as DAPI, quinacrine mustard and quinacrine dihydrochloride have been used in the same technique but none of the above fluorochromes perform as well as the Hoechst stain with respect to fluorescent effect, slow quenching and minimum background fluorescence. 3. Components A. Hoechst Stain (3030145) Hoechst Compound #33258 0.5 μg/ml Hoechst stain contains Thimerosal at 0.1% Dilute 1:10 into 1X Hanks Balanced Salt Solution to stain cells. It may be necessary to titrate the stain to obtain the optimal fluorescence. See notes below in Staining section. B. Mounting Medium (3030345) Citric Acid Monohydrate 4662 mg/L NaHPO 8247 mg/L 4 Glycerol 500 ml Water to 1000 ml C. 10X Hanks Balanced Salt Solution (HBSS) Without Phenol Red and Sodium Bicarbonate (3030245) CaCl .2H O 1855 mg/L 2 2 Glucose 10000 mg/L KCl 4000 mg/L KH PO 600 mg/L 2 4 MgSO .7H O 2000 mg/L 4 2 NaCl 80000 mg/L NaH PO 475 mg/L 2 4 Water to 1000 ml (Caution: Dilute HBSS 1:10 for use with the Hoechst Stain Kit) D. Fixed Control Slide Kit (3030400) contains 5 slides, each with: 1. Vero Cells (negative) 2. Vero Cell infected with Mycoplasma hyorhinis 3. Mouse-mouse hybridoma (negative) 4. Mouse-mouse hybridoma cells infected with Acholeplasma laidlawii 4. Potency The Hoechst Stain should give maximum fluorescent intensity when diluted 1:10 with Hanks Balanced Salt Solution, pH 7.0, achieving a final concentration at 0.05 ug/ml. A lesser or greater dilution may be necessary depending on the microscope being utilized. It is suggested that the investigators determine their own optimum working dilution by testing various dilutions with the known positive specimens included in the kit. 5. Specificity This stain is highly specific for DNA. It is thought to occupy one of the grooves of the DNA double helix, binding by intercalation. The stain will cause cellular nuclei to fluoresce; however, the other major DNA cytoplasmic components - mitochondria - will not. All prokaryotic organisms (Mycoplasmas, bacteria, yeast, fungi) will give off fluorescence. The identification of the contaminant must be made by noting size, morphology and relationship to the cell. Control Slides Zone 1 Vero cell line, a continuous monkey kidney cell line. Zone shows typical results for genetically stable adherent cells demonstrating uniform nuclear staining with little extranuclear staining. Zone 2 Vero cell line co-cultivated with Mycoplasma hyorhinis represents the ideal picture of a cytoadsorbing pleomorphic mycoplasma contaminating a stable continuous cell line. Zone 3 Mouse-mouse hybridoma, an example of a genetically unstable nonadherent cell line, demonstrates the pleomorphism of nuclear structure and extranuclear staining often observed with hybridomas, tumor-derived cell lines and/or cells growing under non- optimal conditions. Zone 4 Mouse-mouse hybridoma co-cultivated with Acholeplasma laidlawii represents the more frequently encountered situation of having to discern normal nuclear and extranuclear staining from contamination with a pleomorphic, non-cytoadsorbing mycoplasmal species. One of the main benefits of this assay procedure is that the stain is not specific only to mycoplasmas, but is a rapid screening procedure for any contaminant. So far all mycoplasma species and strains tested were easily demonstrated by this method. 6. Warning This product is intended for in vitro diagnostic use, but is not limited to this use. The Hoechst-compound is known to bind to DNA by intercalation and is not easily removed. Therefore, care must be taken to avoid direct contact or ingestion of the stain. 7. Storage Concentrated Hoechst stain, Hanks Balanced Salt Solution, slides and mounting medium may be stored at 2oC to 8oC or below. 8. Stability See expiration date on vial for the stain and on slide box for the slides. Dilute stain (1:10 dilution) should be stored at 2oC to 8oC and must not be kept longer than three weeks. 9. Materials Required but not Provided 1. Sterile distilled water for dilution of 10X HBSS. 2. Incubator (37oC) 3. Refrigerator 4. Various glassware 5. Fluorescent microscope 6. Carnoy's fixative or 1 part glacial acetic acid to 3 parts absolute methanol. 10. Procedure A. Growth of Cells Cells should be grown on slides or coverslips in petri dishes or Leighton tubes as desired. The conditions for growth (media, temperature, atmosphere, etc.) should be followed as indicated by the cell line. The cells should be allowed to grow to 50-80% confluency (It is difficult to interpret results in confluent monolayers). It is an excellent idea to plant and stain a slide or coverslip culture with each split of the cell line, thus maintaining an ongoing evaluation of the cell line for mycoplasma, bacteria, yeast and other prokaryotes. B. Staining Note 1: The control slides (#3030400) are already fixed, but unstained, so you may skip over steps 2-5 below. Also please see #4 under Precautions section. Note 2: It is recommended that the stain be used at a dilution of 1:10 of the stock solution (0.05 ug/ml) but can be titrated to determine the optimal fluorescence which may range from 0.05 to 0.5 ug/ml. Note 3: The Hank's Balanced Salt Solution is provided in a 10X concentrate. It must first be diluted with sterile distilled water to a 1X solution for use in step #1. 1. Add the prepared 1x HBSS to the Hoechst Stain solution, to prepare the proper stain dilutions as determined in Note #2. Mix together thoroughly and allow to warm to room temperature. 2. To prepare your cells, first aspirate or remove by pipette almost all of the medium from the petri dish or Leighton tube, leaving only enough medium to barely cover the coverslip. 3. Add a sufficient amount of Carnoy's Fixative (1 part glacial acetic acid to 3 parts absolute methanol) to cover the slide sufficiently (approximately a volume equal to the amount of medium left in the petri dish or Leighton tube) and allow the slide/coverslip to fix for 5 minutes. 4. Remove the medium/fixative and repeat fixation procedure with Carnoy's Fixative for an additional 10 minutes. 5. Air dry the slide/coverslip for 30 minutes. 6. Add 1.0 ml of the working dilution of stain (prepared in step #1) to the slide/coverslip and allow staining to continue for 10-30 minutes at room temperature. (Keep the slide/coverslip covered to prevent dehydration). 7. Remove the stain. 8. Wash the slide/coverslip three times in deionized or distilled water at room temperature allowing 1 minute soaking time per wash. (Do not use salt solutions). 9. Allow the slide/coverslip to air dry. C. Mounting 1. If cells are grown on a SLIDE - Apply a small amount of mounting medium (product #3030345) on the upper-cell sheet surface of the slide and cover with a coverslip. 2. If cells are grown on a COVERSLIP - Add 1 or 2 drops of the mounting medium to a glass slide, put the coverslip on the mounting medium surface side up. Add 1 or 2 drops of additional mounting medium to the upper (cell surface) side of the coverslip and cover with a clean coverslip. 11. Interpretation of Results Specimens should be observed by fluorescent microscopy at between 400-1000X with oil immersion as described below: FILTERS - the choice of a fluorescent microscope is usually determined by budget or what is available. Hoechst Stain #33258 complexed to DNA is excited at 360 nm and emits at 490-500 nm; therefore, barrier exciter filters must be used which fall within these limits. A negative culture will show only nuclear staining (not that mitochondrial DNA will not be demonstrated by this staining technique). Occasionally micronuclei or other nuclear fragments from dead or disrupted cells will appear as spherical bodies. They may be distinguished from Mycoplasma by their large size and brighter fluorescence. A positive cell culture will show the cellular nuclei surrounded by small pinpoint dots of fluorescence, either aggregated in clusters or rows. They will have a uniform size ranging from 0.1 - 0.3 um in diameter and may be pleomorphic. The morphology may range from spherical bodies to filamentous-like forms. Bacteria, yeast and other prokaryotes will show typical size, morphology and growth characteristics (i.e., chains, budding, mycelia, etc.). 12. Limitation of the Test The references listed should be consulted for further information.
Load more

Recommended publications
  • The Nuclear Membrane Determines the Timing of DNA Replication in Xenopus Egg Extracts Gregory H
    The Nuclear Membrane Determines the Timing of DNA Replication in Xenopus Egg Extracts Gregory H. Leno and Ronald A. Laskey Cancer Research Campaign Molecular Embryology Group, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, England Abstract. We have exploited a property of chicken scribed in cultured cells. In contrast to the asynchro- erythrocyte nuclei to analyze the regulation of DNA nous replication seen :between individual nuclei, repli- replication in a cell-free system from Xenopus eggs. cation within multinuclear aggregates was syn- Many individual demembranated nuclei added to the chronous. There ;was a uniform distribution and extract often became enclosed within a common nu- similar fluorescent intensity of the replication loci clear membrane. Nuclei within such a "multinuclear throughout all the nuclei enclosed within the same aggregate" lacked individual membranes but shared the membrane. However, different multinuclear aggregates perimeter membrane of the aggregate. Individual replicated out of synchrony with each other indicating nuclei that were excluded from the aggregates initiated that each membrane-bound aggregate acts as an in- DNA synthesis at different times over a 10-12-h dividual unit of replication. These data indicate that period, as judged by incorporation of biotinylated the nuclear membrane defines the unit of DNA repli- dUTP into discrete replication foci at early times, fol- cation and determines the timing of DNA synthesis in lowed by uniformly intense incorporation at later egg extract resulting in highly coordinated triggering times. Replication forks were clustered in spots, rings, of DNA replication on the DNA it encloses. and horsesh0e-shaped structures similar to those de- NITIATION of DNA replication occurs at thousands of defines the unit of DNA replication in the egg extract.
    [Show full text]
  • Ab228551 Hoechst 33342 Staining Dye Solution
    Version 1 Last updated 12 March 2018 ab228551 Hoechst 33342 Staining Dye Solution For labeling DNA in fluorescence microscopy. This product is for research use only and is not intended for diagnostic use. Copyright © 2017 Abcam. All rights reserved Table of Contents 1. Overview 1 2. Materials Supplied and Storage 3 3. Materials Required, Not Supplied 4 4. General guidelines, precautions, and troubleshooting 5 5. Reagent Preparation 6 6. Assay Procedure 7 7. FAQs / Troubleshooting 8 8. Notes 9 Copyright © 2017 Abcam. All rights reserved 1. Overview Hoechst 33342 Staining Dye Solution (ab228551) is a fluorescent stain for labeling DNA in fluorescence microscopy. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. It can also be used to detect the contents of a sample DNA by plotting a standard emission-to content curve. Figure 1. Chemical structure of Hoechst 33342. Figure 2. Spectrum of Hoechst 33342. ab228551 Hoechst 33342 Staining Dye Solution 1 Pellet cells by centrifugation. Resuspend the cells in buffered salt solutions or media, with optimal dye binding at pH 7.4. Add Hoechst stain using concentrations between 0.5 and 5 µM and incubate for 15 to 60 minutes. ab228551 Hoechst 33342 Staining Dye Solution 2 2. Materials Supplied and Storage Store kit at -20°C in the dark immediately on receipt and check below for storage for individual components. Kit can be stored for 1 year from receipt, if components have not been reconstituted. Storage temperatur Item Quantity e (before prep) Hoechst 33342 5 mL -20°C (20 mM Solution in Water) ab228551 Hoechst 33342 Staining Dye Solution 3 3.
    [Show full text]
  • An Acromegaly Disease Zebrafish Model Reveals Decline in Body
    biology Article An Acromegaly Disease Zebrafish Model Reveals Decline in Body Stem Cell Number along with Signs of Premature Aging Abdalla Elbialy 1,2 , Yoji Igarashi 1 , Shuichi Asakawa 1, Shugo Watabe 3 and Shigeharu Kinoshita 1,* 1 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8654, Japan; [email protected] (A.E.); [email protected] (Y.I.); [email protected] (S.A.) 2 Laboratory of Fish Diseases, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, Egypt 3 School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa 252-0313, Japan; [email protected] * Correspondence: [email protected] Received: 27 April 2020; Accepted: 4 June 2020; Published: 7 June 2020 Abstract: In our previous publication, it was shown that growth hormone (GH) excess in acromegaly affects the cell integrity of somatic cells through increased DNA damage throughout the body and impaired DNA repair pathways. Acromegaly is a hormone disorder pathological condition that develops as a result of growth hormone over-secretion from the pituitary gland. We produced a zebrafish acromegaly model to gain a better understanding of the excess GH effects at the cellular level. Here we show that the acromegaly zebrafish model progressively reduced the number of stem cells in different organs and increased oxidative stress in stem cells. Importantly, the decline in the stem cells was even more apparent than in aged fish. The controversy and debate over the use of GH as an anti-aging therapy have been going on for several years.
    [Show full text]
  • And Poly(Vinyl Chloride) Nanoparticles with BHK-21 Cell Line
    www.nature.com/scientificreports OPEN Understanding the interactions of poly(methyl methacrylate) and poly(vinyl chloride) nanoparticles with BHK‑21 cell line Gomathi Mahadevan & Suresh Valiyaveettil * Microplastic and nanoplastic particles are prevalent in the environment and are beginning to enter the living system through multiple channels. Currently, little is known about the impact of plastic nanoparticles in living organisms. In order to investigate the health impact of micro‑ and nanoparticles of common polymers in a systematic way, luminescent plastic nanoparticles from two common polymers, polyvinyl chloride (PVC) and poly (methyl methacrylate) (PMMA) with relatively narrow size distribution are prepared using a nanoprecipitation method. As a model system, BHK‑21 cells were exposed to polymer nanoparticles to understand the mode of uptake, internalization and biochemical changes inside the cells. The cellular efects of the nanoparticles were evaluated by monitoring the changes in cell viability, cell morphology, concentrations of reactive oxygen species (ROS), adenine triphosphate (ATP) and lactate dehydrogenase at diferent concentrations of the nanoparticles and time of exposure. PVC and PMMA nanoparticles induced a reduction in the cell viability along with a reduction of ATP and increase of ROS concentrations in a dose‑ and time‑dependent manner. The plastic nanoparticles are internalized into the cell via endocytosis, as confrmed by Dynasore inhibition assay and colocalization with latex beads. Our fndings suggest that plastic nanoparticle internalization could perturb cellular physiology and afect cell survival under laboratory conditions. Plastic waste accumulated in the environment undergoes slow degradation and disintegration under the ambient conditions and in presence of sunlight to generate smaller fragments called microplastics (size below 5 mm) or nanoplastics (below 1 µm)1,2.
    [Show full text]
  • Towards the Development of a Novel DNA Binding Fluorescent Cell Stain;
    Running head: FLUORESCENT DNA BINDING CELL STAINS 1 Towards the Development of a Novel DNA Binding Fluorescent Cell Stain; An Analysis of Common DNA Dyes and Their Applications Lauren Kapteyn A Senior Thesis submitted in partial fulfillment of the requirements for graduation in the Honors Program Liberty University Spring 2018 FLUORESCENT DNA BINDING CELL STAINS 2 Acceptance of Senior Honors Thesis This Senior Honors Thesis is accepted in partial fulfillment of the requirements for graduation from the Honors Program of Liberty University. ______________________________ Michael Korn, Dr. rer. nat. Thesis Chair ______________________________ Jeremy Sellers, Ph.D. Committee Member ______________________________ David Dinsmore, Ed. D., P.E. Committee Member ______________________________ Christopher Nelson, M.F.A. Assistant Honors Director ______________________________ Date FLUORESCENT DNA BINDING CELL STAINS 3 Abstract Fluorescent deoxyribonucleic acid (DNA) stains that permeate cells are used to observe cellular processes in vivo, making them valuable tools. For the proposal of a new stain, ethidium bromide, cyanine dyes, Hoechst stains, macarpine, DAPI and DRAQ5 are first examined. Features contributing to DNA binding mechanism, toxicity, and cell membrane transport are analyzed for these compounds. The mechanism of DNA binding contributes to the toxicity of the compound. For dyes with well-understood transport mechanisms, multi-drug protein transporters are vital players, though this remains an area for ongoing research as many mechanisms are not well studied. A novel anthraquinone- based compound is proposed and the DNA binding mechanism, cell permeability and fluorescence are predicted based on the traits of existing dyes. FLUORESCENT DNA BINDING CELL STAINS 4 Towards the Development of a Novel DNA Binding Fluorescent Cell Stain An Analysis of Common DNA Dyes and Their Applications Background Fluorescent molecules (fluorophores) are widely used in the biological sciences to visualize cells and subcellular structures.
    [Show full text]
  • Flow Cytometry of Apoptosis UNIT 18.8
    Flow Cytometry of Apoptosis UNIT 18.8 This unit describes the most common methods applicable to flow cytometry that make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. An early event of apoptosis, dissipation of the mitochondrial transmembrane potential, can be measured using a number of fluorochromes that are sensitive to the electrochemical potential within this organelle (see Basic Protocol 1). Another early event of apoptosis, caspase activation, can be measured either directly, by immunocytochemical detection of the epitope that characterizes activated caspase (see Basic Protocol 2), or indirectly by immunocyto- chemical detection of the caspase-3 cleavage product, the p85 fragment of poly(ADP-ri- bose) polymerase (see Basic Protocol 4). Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V (annexin V–FITC); this assay is combined with analysis of the exclusion of the plasma membrane integrity probe propidium iodide (PI; see Basic Protocol 5). Also described are methods of analysis of DNA fragmentation based either on DNA content of cells with fractional (sub-G1) DNA content (see Basic Protocol 6 and Alternate Protocol 1) or by DNA strand-break labeling (Terminal deoxynucleotidyltransferase–mediated dUTP Nick End Labeling, TUNEL; or In Situ End Labeling, ISEL; see Basic Protocol 7). Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that cross-links protein and thereby makes them less immunogenic.
    [Show full text]
  • Design, Visualize and Detect
    Thermo Scientific Pierce Antibody Immunostaining Guide Design, visualize and detect Immunostaining Primary Antibodies • Secondary Antibodies • Staining Dyes • Kits Thermo Scientific Table of Contents Pierce Antibody Immunostaining Guide Page Introduction 1-3 The Cell 4-5 Mammalian Cell Type Choices 6-8 Immunohistochemistry 9-12 Immunofluorescence 13-22 Secondary Antibodies 23-27 Primary Antibodies by Cellular Structures 28-31 by Research Areas 32-43 by Cell Signaling 44-59 by Biological Processes 60-76 Left: Detection of mouse anti-α-tubulin in an A549 cell in Telophase with Thermo Scientific DyLight Dye 550-GAM. Chromosomes (orange) at the poles become Introduction diffuse, while nuclei (blue) divide into two future cells. Immunofluorescence (IF) and immunohistochemistry (IHC) are two methods commonly used to detect proteins in a cellular context. Immunofluorescent detection of proteins can be performed on both fixed cells in culture and on paraffin or frozen tissue sections. The advantages of using IF to detect cellular proteins includes the ability to visualize the subcellular location of protein(s) of interest, assess both protein expression and post-translational modifications, and design multiplex experiments. When IF detection is extended to tissues sections (IHC), a higher level of resolution is achieved because researchers are analyzing target protein(s) in a near physiological state, making it ideal for assessing normal and disease tissues. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1 Need Antibodies? Build a Better Antibody Introduction We have over 30,000 antibodies in 42 research areas. Use our custom services to produce antibodies you can trust.
    [Show full text]
  • A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    cells Article A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy Anna Ligasová 1,* , Markéta Vydržalová 2, Renata Buriánová 1, Lenka Br ˚uˇcková 2, Renata Veˇceˇrová 3, Anna Janošt’áková 1 and Karel Koberna 1,* 1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hnˇevotínská 5, 779 00 Olomouc, Czech Republic; [email protected] (R.B.); [email protected] (A.J.) 2 Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic; [email protected] (M.V.); [email protected] (L.B.) 3 Department of Microbiology, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hnˇevotínská 3, 779 00 Olomouc, Czech Republic; [email protected] * Correspondence: [email protected] (A.L.); [email protected] (K.K.); Tel.: +420-585632184 (A.L.); +420-585632184 (K.K.) Received: 9 October 2019; Accepted: 23 November 2019; Published: 25 November 2019 Abstract: Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas’ DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas.
    [Show full text]
  • Hoechst 33342
    Hoechst 33342 CATALOG #639 RESEARCH USE ONLY INTRODUCTION HOW TO USE Hoechst 33342 is a popular cell-permeant, blue fl uores- Hoechst 33342 is supplied ready-to-use at 200 cent nuclear stain. It is used to visualize the nuclei of μg/mL. To stain cellular nuclei: living or fi xed cells and tissues and is often used to distin- 1. Add Hoechst 33342 to the cell sample media guish condensed, pyknotic nuclei in apoptotic cells. at 0.5% v/v. For example, add 1.5 μL Hoechst to 300 μL of cells. Hoechst 33342 emits blue fl uorescence when bound to 2. Incubate 10-20 minutes at room temperature. double stranded DNA. It is slightly more membrane per- 3. Visualize with a fl uorescence microscope by meant than the Hoechst 33258 analog. Hoechst 33342 using a UV excitation and blue emission fi lter. may be used to identify healthy or apoptotic nuclear mor- The blue Hoechst stain fl uoresces at 461 nm. phology and for cell cycle studies. 4. Alternatively, cells may be analyzed with a Each vial of Hoechst 33342 contains 1 mL of aqueous fl ow cytometer using a UV excitation source. solution at 200 μg/mL (catalog #639). It is ready to use: 5. When bound to dsDNA, the maximum absorp- just add it to the cell culture media at 0.5%, incubate 10- tion is 350 nm and the maximum emission is 20 minutes, and analyze. Hoechst 33342 can be excited 461. with a xenon or mercury-arc lamp or with a UV laser, and may be used in fl ow cytometry systems utilizing UV excita- SPECIFICATIONS tion sources.
    [Show full text]
  • BD Pharmingen™ Cell Cycle
    BD Pharmingen™ Bioimaging Certified Reagent Technical Data Sheet Cell Cycle Kit Product Information Catalog Number: 558662 Size: 100 tests Please Note: For appropriate safety and disposal guidelines, refer to the MSDS. The kit contains two parts, A and B, which are shipped separately. Confocal image, using the BD Pathway™ 435 Bioimaging system and a 20x (0.75 NA) objective, of HeLa cells that were stained with the three kit components, Alexa Fluor® 488 Mouse anti-BrdU (pseudo-colored green), Alexa Fluor® 647 Rat anti-Histone H3 (pS28) (pseudo-colored red) and Hoechst 33342 (pseudo-colored blue). Co- staining of Hoechst 33342 and Histone H3 (pS28) appears pink. Kit Contents and Storage Upon arrival store Parts A and B as follows: Part A, store at 4oC: Description # of vials Component No.* Bioimaging Certified Alexa Fluor® 488 Mouse anti-BrdU 1 51-9004981 Bioimaging Certified Alexa Fluor® 647 Rat anti-Histone H3 (pS28) 1 51-9004980 5x Fixation Buffer 1 51-9005210 Perm Buffer III 1 51-9004976 Stain Buffer (FBS) 1 51-9004979 PBS (10X) Concentrate 1 51-9004978 Hoechst 33342 Solution 1 51-9004975 Part B, store at -80oC: Description # of vials Component No.* DNase 5 51-2358KC BrdU 1 51-2420KC * Component numbers are provided for identification only. The components may not be ordered individually. 558662 Rev. 2 1 of 8 Introduction The cell cycle consists of a series of processes involved in cell growth and replication. This coordinated division of cells can be separated into two major stages, interphase (the phase between mitotic events) and mitosis. There are three distinct, successive stages within interphase, namely G1, S, and G2 phases.
    [Show full text]
  • AUTHENTICATION and CHARACTERIZATION of ANIMAL CELL LINES: TOWARDS BEST PRACTICES in CELL CULTURE Yvonne A
    AUTHENTICATION AND CHARACTERIZATION OF ANIMAL CELL LINES: TOWARDS BEST PRACTICES IN CELL CULTURE Yvonne A. Reid, Ph.D. Manager/Scientist, Cell Biology Program, ATCC February 27th 2014 ATCC • Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA • ATCC serves and supports the scientific community with industry-standard products and innovative solutions • World’s leading biological resource center and provider of biological standards • Broad range of biological materials . Microorganisms . Cell lines . Derivatives . Bioproducts 2 OutlineMinimum Requirements for Reproducible Results Monitoring cell morphology Optimizing cell growth Applying the ‘Seed Stock’ concept Using cells at low passage or PDL Checking for microbial contamination Confirming species (misidentification or cross-contamination) Confirming characteristics/functionality History of misidentified cell lines – impact on research Reducing cellular and microbial contamination Receiving a cell line into the laboratory 3 Monitoring cell morphology Fibroblast-like Lymphoblastoid-like Epithelial-like Senescing cells Adherent Suspension Adherent β- galactosidase • Monitor morphology: shape, membrane structure, optical properties – at various stages of cell growth . Detecting senescing cells: larger, vacuolated, more heterogeneous . Detecting overt microbial contamination 4 Optimizing cell growth Growth profile 1.00E+05 PDT:33 hr • Recommended inoculum 2 PDT:21 hr • Population Doubling Time (PDT) 1.00E+04 • Optimum concentration range for Cells/cm PDT:20
    [Show full text]
  • The Multidrug Resistance Transporter ABCG2 (Breast Cancer Resistance Protein 1) Effluxes Hoechst 33342 and Is Overexpressed in Hematopoietic Stem Cells
    22 Vol. 8, 22–28, January 2002 Clinical Cancer Research Advances in Brief The Multidrug Resistance Transporter ABCG2 (Breast Cancer Resistance Protein 1) Effluxes Hoechst 33342 and Is Overexpressed in Hematopoietic Stem Cells Min Kim, Heth Turnquist, John Jackson, porter and that ABCG2/Bcrp1 expression may serve as a Magda Sgagias, Ying Yan, Maokai Gong, marker for hematopoietic stem cells in hematopoietic cells. Michael Dean, John G. Sharp, and Introduction Kenneth Cowan1 The emergence of drug-resistant tumor cells remains a Eppley Institute for Cancer Research [M. K., H. T., M. S., Y. Y., major problem in cancer chemotherapy. The development of K. C.], Department of Pathology [J. J.], and Department of Cell Biology [J. G. S.], University of Nebraska Medical Center, Omaha, multidrug resistance is often characterized by reduced intracel- Nebraska 68198, and National Cancer Institute, Bethesda, Maryland lular levels of many diverse classes of cytotoxic drugs and is 20892 [M. G., M. D.] frequently associated with overexpression of one or more mem- bers of the ABC2 superfamily of membrane transporters (1). These transporters include P-glycoprotein, encoded by the Abstract MDR1 gene (2), and the multidrug resistance-associated protein The human ATP-binding cassette superfamily G (MRP) gene (3). Another MDR gene has been described re- (White) member 2 (ABCG2) gene and its murine homologue cently and referred to as the BCRP gene. The BCRP gene is an breast cancer resistance protein 1 (Bcrp1) are recently de- ABC half-transporter that is associated with resistance to mi- scribed ATP-binding cassette transporters associated with toxantrone, anthracyclines, and camptothecins (4–6).
    [Show full text]